Spatial mapping of cellular senescence: emerging challenges and opportunities

In the paper

“As an alternative to antibody-based methods, innovations for unbiased mass spectrometry-based studies are evolving to query a larger swath of the proteome and metabolome with spatial orientation in tissues, although these methods are still fairly limited in throughput and resolution43,44, restricting their application to mapping rare senescent cells.”

What the sources say

• Disputed

  Split into 2 atoms — 0 confirmed, 1 partially supported, 1 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Checked by a reasoning model — may be wrong.

  cites [43] Taylor, M. J., Liyu, A., Vertes, A. & Anderton, C. R. Ambient single-cell analysis and native tissue imaging using laser-ablation electrospray ionization mass spectrometry with increased spatial resolution. J. Am. Soc. Mass Spectrom. 32, 2490–2494 (2021). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [44] Buczak, K. et al. Spatially resolved analysis of FFPE tissue proteomes by quantitative mass spectrometry. Nat. Protoc. 15, 2956–2979 (2020). source

In the paper

“For example, the commonly used marker senescence-associated β-galactosidase (SA-β-Gal) at pH 6 can be detected in vitro in confluent and immortalized cells6 and in activated macrophages7, while cell cycle kinase inhibitors p16INK4a and p21CIP1 can be expressed irrespectively of senescence in certain contexts7,8.”

What the sources say

• ?Not checked — no full text of the sourcecites [6] Severino, J., Allen, R. G., Balin, S., Balin, A. & Cristofalo, V. J. Is β-galactosidase staining a marker of senescence in vitro and in vivo? Exp. Cell Res. 257, 162–171 (2000). source
• Partly supported

  The Results (Nature of cells attracted by SCs into peritoneal cavity; page unavailable) report elevated β-galactosidase activity at pH 6 in macrophages elicited by senescent cells. That supports detection in some macrophages, but the reported macrophage finding is in mice, not an in-vitro demonstration. The Discussion (page unavailable) also cautions that β-galactosidase activity does not increase with every type of macrophage activation. The confluent and immortalized cell clause is attached to a different citation and is not judged here. (LLM, conf=0.83)

  cites [7] Hall, B. M. et al. Aging of mice is associated with p16Ink4a- and β-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells. Aging 8, 1294–1315 (2016). sourceExperimental — accuracy not yet measured

In the paper

“It should be noted, however, that senescent cells also have important physiological roles and that their clearance may be detrimental in certain conditions3–5.”

What the sources say

• Supported

  The Results section, “Elimination of Senescent Cells Delays Cutaneous Wound Healing” (page not supplied), reports that senescent cells facilitate wound repair in mice and that their absence significantly delays wound closure. This supports both a beneficial physiological role and the hedged claim that clearance may be detrimental in certain conditions—specifically, during cutaneous wound healing. (LLM, conf=0.93)

  cites [3] Demaria, M. et al. An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev. Cell 31, 722–733 (2014). source
• Supported

  The Introduction describes senescence as a tumor-suppressive mechanism, supporting a physiological role. The Clinical Perspective reports that senescent-cell elimination altered pulmonary hemodynamics in healthy animals and exacerbated experimental pulmonary hypertension. The supplied passages do not provide page numbers. (LLM, conf=0.92)

  cites [4] Born, E. et al. Eliminating senescent cells can promote pulmonary hypertension development and progression. Circulation 147, 650–666 (2023). source
• Supported

  In “p16INK4a+ fibroblasts promote epithelial stem cell regeneration” (page unavailable), the paper reports that senolytic treatment after lung injury reduced p16INK4a+ cells and significantly impaired airway-cell regeneration. The Discussion (page unavailable) describes a reparative role for these fibroblasts, some of which have senescent characteristics. This supports the qualified claim that clearance may be detrimental in certain conditions. (LLM, conf=0.88)

  cites [5] Reyes, N. S. et al. Sentinel p16INK4a+ cells in the basement membrane form a reparative niche in the lung. Science 378, 192–201 (2022). source

In the paper

“Senescent cells can induce senescence in neighboring cells14,15 and, through the SASP, recruit immune cells to their vicinity.”

What the sources say

• Supported

  The citation is attached to the claim that senescent cells induce senescence in neighboring cells. In “Paracrine senescence is a stable arrest mediated by secreted factors” (page not provided), nearby normal cells showed reduced proliferation after oncogene-induced senescence, and the authors conclude that senescence can be transmitted by soluble factors. The immune-cell recruitment clause is not attached to this citation. (LLM, conf=0.94)

  cites [14] Acosta, J. C. et al. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat. Cell Biol. 15, 978–990 (2013). source
• Supported

  The citation follows the claim that senescent cells can induce senescence in other cells; the immune-cell recruitment clause comes after it. In the Results section on senescent-cell transplantation, the paper reports increased senescence markers in recipient mice’s own adipose-tissue cells after senescent cells were transplanted. (LLM, conf=0.91)

  cites [15] Xu, M. et al. Senolytics improve physical function and increase lifespan in old age. Nat. Med. 24, 1246–1256 (2018). source

In the paper

“Expression of p21CIP1, p16INK4A, p53, components of the DNA damage response (DDR; for example, γH2A.X and 53BP1)22, decreased lamin B1 (ref. 23), loss of nuclear high-mobility group box 1 (HMGB1)24 and expression of GLB1 (ref. 25) (which encodes lysosomal β-d-galactosidase) are common senescence-associated markers assessed in tissues by immunohistochemistry and immunofluorescence.”

What the sources say

• ?Not checked — no full text of the sourcecites [22] Wang, C. et al. DNA damage response and cellular senescence in tissues of aging mice. Aging Cell 8, 311–323 (2009). source
• Supported

  Split into 2 atoms — 2 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [23] Freund, A., Laberge, R. M., Demaria, M. & Campisi, J. Lamin B1 loss is a senescence-associated biomarker. Mol. Biol. Cell 23, 2066–2075 (2012). source
• Supported

  Split into 2 atoms — 2 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [24] Davalos, A. R. et al. p53-dependent release of alarmin HMGB1 is a central mediator of senescent phenotypes. J. Cell Biol. 201, 613–629 (2013). source

In the paper

“For example, structured illumination microscopy was used to estimate the density of nuclear pores33.”

What the sources say

• Supported

  The Materials and methods section (‘SIM and measurement of nuclear pore density’) says the authors imaged cells by 3D structured illumination microscopy and manually counted individual nuclear pore complexes in the images. Figure 1F–G reports the resulting density measurements in pores per square micrometer. (LLM, conf=0.95)

  cites [33] Boumendil, C., Hari, P., Olsen, K. C. F., Acosta, J. C. & Bickmore, W. A. Nuclear pore density controls heterochromatin reorganization during senescence. Genes Dev. 33, 144–149 (2019). source

In the paper

“Advantages of this method are that it can be applied to FFPE tissues and combined with other immunohistochemical techniques.”

What the sources say

• Supported

  The Discussion states that Sudan Black B staining detects lipofuscin in FFPE tissue sections. The Results report concomitant staining with Sudan Black B and the proliferation marker Ki67 in cultured cells, supporting compatibility with an immunostaining technique; the paper does not demonstrate that combination in FFPE sections. (LLM, conf=0.86)

  cites [21] Georgakopoulou, E. A. et al. Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues. Aging 5, 37–50 (2013). source

In the paper

“These methods can be applied to both frozen and FFPE tissue sections.”

What the sources say

• Partly supported

  The Methods > RNA-ISH section describes applying RNAscope to paraffin tissue sections, supporting the FFPE portion of the claim. It does not describe RNA-ISH on frozen sections; searches for frozen sections and FFPE found no such passage. Page numbers are not available in our copy. (LLM, conf=0.87)

  cites [13] Ogrodnik, M. et al. Whole-body senescent cell clearance alleviates age-related brain inflammation and cognitive impairment in mice. Aging Cell 20, e13296 (2021). sourceExperimental — accuracy not yet measured
• Partly supported

  The Materials and Methods section (page unavailable) describes RNA-ISH on paraffin tissue sections, supporting its use on FFPE sections. It does not describe RNA-ISH on frozen sections; the frozen liver tissue mentioned elsewhere was reserved for biochemical analyses. (LLM, conf=0.88)

  cites [16] Lagnado, A. et al. Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner. EMBO J. 40, e106048 (2021). sourceExperimental — accuracy not yet measured
• Partly supported

  The STAR★Methods section on RNA in situ hybridization describes applying RNA-ISH to paraffin sections. It does not describe RNA-ISH on frozen sections, and it does not explicitly say the paraffin sections were formalin-fixed, so the claim about both frozen and FFPE tissue is not fully supported. (LLM, conf=0.84)

  cites [31] Ogrodnik, M. et al. Obesity-induced cellular senescence drives anxiety and impairs neurogenesis. Cell Metab. 29, 1061–1077 (2019). sourceExperimental — accuracy not yet measured

In the paper

“Super-resolution microscopy has emerged as having great potential to investigate biological samples at unprecedented levels of detail, as new and powerful fluorescence-based methods now permit imaging cellular compartments at a resolution beyond that of the diffraction limit of light (<200 nm).”

What the sources say

• Supported

  The Abstract says far-field super-resolution techniques overcome the diffraction limit. The Resolution section reports achievable resolutions down to 120 nm and describes their use to examine mitochondrial morphology and membrane domains (page numbers not provided). (LLM, conf=0.92)

  cites [32] Valli, J. et al. Seeing beyond the limit: a guide to choosing the right super-resolution microscopy technique. J. Biol. Chem. 297, 100791 (2021). source

In the paper

“Thorough in situ characterization of DDR foci also requires high-resolution imaging to detect and quantify distinct intranuclear puncta, as DDR foci are often under 1 μm34,35.”

What the sources say

• Partly supported

  The Introduction says DNA damage foci can be detected in cell nuclei by fluorescence microscopy, and the Results describe monitoring and distinguishing small from enlarged foci. However, our copy does not establish that high-resolution imaging is required or that DDR foci are often under 1 μm (searched for size, resolution, and imaging requirements). (LLM, conf=0.78)

  cites [34] Rodier, F. et al. DNA-SCARS: distinct nuclear structures that sustain damage-induced senescence growth arrest and inflammatory cytokine secretion. J. Cell Sci. 124, 68–81 (2011). sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion (page unavailable) says super-resolution STED microscopy improved the resolution of clustered telomere signals, supporting the need for higher-resolution imaging to distinguish nearby signals. It does not establish that DDR foci are often under 1 μm or that this size is why high-resolution imaging is required for their in situ characterization. (LLM, conf=0.83)

  cites [35] Anderson, R. et al. Length-independent telomere damage drives post-mitotic cardiomyocyte senescence. EMBO J. 38, e100492 (2019). sourceExperimental — accuracy not yet measured

In the paper

“For example, HMGB1, a ubiquitous and predominantly nuclear protein, localizes to the cytoplasm and is secreted during senescence24.”

What the sources say

• Supported

  The Introduction describes HMGB1 as an abundant protein, and the Results (“Senescent human cells secrete HMGB1”) show that it is abundant in nonsenescent nuclei but depleted from senescent nuclei, with some cytoplasmic staining. That section also detects HMGB1 in conditioned media; “Senescent cells actively export nuclear HMGB1” confirms its export from the nucleus. (LLM, conf=0.88)

  cites [24] Davalos, A. R. et al. p53-dependent release of alarmin HMGB1 is a central mediator of senescent phenotypes. J. Cell Biol. 201, 613–629 (2013). source

In the paper

“Already there are reports of Visium being used to investigate the spatial characteristics of cellular senescence in mouse52 and human brains53 as well as to decipher age-dependent tissue-regeneration capabilities54.”

What the sources say

• Supported

  The citation to this paper is attached to the mouse-brain part of the sentence, not the separate claims about human brains or tissue regeneration. The Introduction describes mapping senescence-associated microdomains in young and aged mouse brains, and “Tissue processing and RNA sequencing” states that the brain sections were placed on Visium Spatial Gene Expression Slides. Page numbers were not available in our copy. (LLM, conf=0.94)

  cites [52] Kiss, T. et al. Spatial transcriptomic analysis reveals inflammatory foci defined by senescent cells in the white matter, hippocampi and cortical grey matter in the aged mouse brain. GeroScience 44, 661–681 (2022). source
• Supported

  Citation 53 supports the human-brain portion of the sentence. The Results describe using a 10x Genomics Visium spatial transcriptomic dataset of postmortem human brain to map the spatial localization of a senescence-gene-enriched module; the mouse-brain and tissue-regeneration claims have separate citations. (LLM, conf=0.94)

  cites [53] Xu, P. et al. The landscape of human tissue and cell type specific expression and co-regulation of senescence genes. Mol. Neurodegener. 17, 5 (2022). source
• Supported

  The Methods state that the study used the Visium Spatial Gene Expression System. The Discussion reports spatial transcriptomic analysis of impaired digit regeneration in aged mice and a partial rescue of the age-dependent decline. Citation 54 supports the regeneration clause; the brain-senescence clauses have separate citations. (LLM, conf=0.94)

  cites [54] Tower, R. J. et al. Spatial transcriptomics reveals metabolic changes underly age-dependent declines in digit regeneration. eLife 11, e71542 (2022). source

In the paper

“The ability of STORM to provide a detailed rendering of the structure of chromatin fibers37 makes it ideal to investigate changes in heterochromatin distribution within senescent nuclei, such as the loss of lamin-associated domains38, which has been primarily investigated via electron microscopy techniques39.”

What the sources say

• Partly supported

  The Results (page not provided) report using electron microscopy to find reduced staining of peripheral heterochromatin in senescent nuclei. The paper does not establish that lamin-associated domains themselves were lost or that their loss has primarily been investigated by electron microscopy. (LLM, conf=0.84)

  cites [39] De Cecco, M. et al. Genomes of replicatively senescent cells undergo global epigenetic changes leading to gene silencing and activation of transposable elements. Aging Cell 12, 247–256 (2013). sourceExperimental — accuracy not yet measured

In the paper

“Similarly, levels of lamin B1, which is a component of the nuclear lamina, decrease during senescence23, and its detection requires sufficient resolution to visualize a 30–100-nm-thick structure.”

What the sources say

• Supported

  The Introduction identifies lamin B1 as a B-type lamin of the nuclear lamina. The Results report that lamin B1 protein levels declined markedly in senescent cells. Citation 23 is attached to the decline during senescence, not to the subsequent statement about resolving a 30–100-nm-thick structure. (LLM, conf=0.93)

  cites [23] Freund, A., Laberge, R. M., Demaria, M. & Campisi, J. Lamin B1 loss is a senescence-associated biomarker. Mol. Biol. Cell 23, 2066–2075 (2012). source

In the paper

“Methods such as cyclic immunofluorescence (CyCIF)45 and iterative indirect immunofluorescence imaging (4i)46, which do not require proprietary instrumentation, allow the elution of antibodies in relatively mild conditions that preserve the sample structure up to and possibly over 20 rounds of elution and imaging, allowing the detection of over 40 protein targets.”

What the sources say

• Partly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [45] Lin, J. R., Fallahi-Sichani, M., Chen, J. Y. & Sorger, P. K. Cyclic immunofluorescence (CycIF), a highly multiplexed method for single-cell imaging. Curr. Protoc. Chem. Biol. 8, 251–264 (2016). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [46] Gut, G., Herrmann, M. D. & Pelkmans, L. Multiplexed protein maps link subcellular organization to cellular states. Science 361, eaar7042 (2018). source

In the paper

“Interestingly, p21CIP1-positive nuclei exhibited higher predicted levels of senescence in multiple tissues, while proliferating cells (indicated by 5-ethynyl-2′-deoxyuridine (EdU) incorporation) showed lower levels65.”

What the sources say

• Supported

  The Results section reports higher predicted senescence for p21Cip1-positive cells in mouse liver and for p21Cip1-positive nuclei in human dermis using the RS and IR models. It also reports that EdU-positive cells had lower senescence scores in mouse skin and testis. (LLM, conf=0.92)

  cites [65] Heckenbach, I. et al. Nuclear morphology is a deep learning biomarker of cellular senescence. Nat. Aging 2, 742–755 (2022). source

In the paper

“Furthermore, PIXEL-seq uses DNA arrays called ‘polony gels’, which capture tissue RNA from a single-cell layer touching the gel, similar to confocal microscopy, and restrain template diffusion, thus improving single-cell resolution63.”

What the sources say

• Supported

  The Results section on the mouse olfactory bulb reports that polony gels appeared to capture RNA from the gel-contacting cell layer rather than deeper tissue. It also reports limited template drift and says gel-based capture increased resolution; the Introduction identifies polony gels as the basis of Pixel-seq. (LLM, conf=0.91)

  cites [63] Fu, X. et al. Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain. Cell 185, 4621–4633 (2022). source

In the paper

“Recently, several ultra-high-resolution technologies such as Seq-Scope61, Stereo-seq62 and polony-indexed library sequencing (PIXEL-seq)63 have been developed, which could prove ideal for identification and characterization of senescent cells and their interactions with the environment. In addition to providing submicrometer resolution, which is comparable to that of optical microscopy, these meth”

What the sources say

• Supported

  The citation identifies Seq-Scope, and the Introduction reports its submicrometer resolution (0.5–0.8 μm) and transcriptome capture output of up to 23–27 UMIs/μm². The Results likewise describe high capture efficiency. This source is cited for Seq-Scope, not for the separately cited technologies. (LLM, conf=0.92)

  cites [61] Cho, C.-S. et al. Microscopic examination of spatial transcriptome using Seq-Scope. Cell 184, 3559–3572 (2021). source
• Supported

  The citation identifies Stereo-seq, which this paper introduces. The methods description (p. 3) reports 220-nm capture spots spaced 500 or 715 nm apart, and the benchmark (p. 3) reports 69 unique molecular identifiers per 2-μm-diameter bin—approximately 22 per μm², consistent with the claim’s approximate figure. The separate citations attached to Seq-Scope and PIXEL-seq are not judged here. (LLM, conf=0.86)

  cites [62] Chen, A. et al. Spatiotemporal transcriptomic atlas of mouse organogenesis using DNA nanoball-patterned arrays. Cell 185, 1777–1792 (2022). source
• Partly supported

  The Introduction identifies this paper as the report of Pixel-seq and describes high-resolution tissue mapping with efficient RNA capture; the mouse olfactory-bulb Results likewise report high capture efficiency. But the Results describe Pixel-seq’s assay feature resolution as 1 μm, while the submicrometer figure refers to gel-to-gel replication. Our copy does not report ~23 unique transcripts per μm² or establish that figure for all three named methods; pages are not provided in the indexed copy. (LLM, conf=0.86)

  cites [63] Fu, X. et al. Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain. Cell 185, 4621–4633 (2022). sourceExperimental — accuracy not yet measured

In the paper

“Deep learning methods have been recently developed to detect senescent cells in culture with phase-contrast imaging99 and tissues with H&E and DAPI staining65.”

What the sources say

• Supported

  The Methods (Microscopic imaging) describe phase-contrast images of cultured control and senescent HUVECs. The Results (High accuracy identification of senescent cells by a CNN) report that the trained network classified senescent and control cells with high accuracy. The H&E and DAPI tissue-imaging clause is attached to a different citation, so it is not attributed to this source. (LLM, conf=0.94)

  cites [99] Kusumoto, D. et al. Anti-senescent drug screening by deep learning-based morphology senescence scoring. Nat. Commun. 12, 257 (2021). source
• Supported

  The Results section (‘Predictor translates across species and tissues in vivo’; page unavailable) reports applying the senescence predictor to DAPI-stained mouse liver sections and H&E-stained liver tissue. The paper also reports its use on H&E-stained human dermis. The phase-contrast imaging clause is attributed to citation 99, not this source. (LLM, conf=0.94)

  cites [65] Heckenbach, I. et al. Nuclear morphology is a deep learning biomarker of cellular senescence. Nat. Aging 2, 742–755 (2022). source

In the paper

“Further analysis of skin biopsies revealed that predicted senescence in the dermis of more than 160 individuals increased with age and showed expected associations with disease outcomes.”

What the sources say

• Supported

  The Results section, “Human dermis shows an increase in senescent nuclei with age,” reports analysis of dermal biopsies from 169 people and an age-dependent increase in predicted senescence. “Senescence is associated with multiple health conditions” reports associations with diagnosed conditions, including fewer malignancies and higher frequencies of osteoporosis and osteoarthritis. The paper notes that several individual-condition associations did not remain significant after multiple-test correction. (LLM, conf=0.91)

  cites [65] Heckenbach, I. et al. Nuclear morphology is a deep learning biomarker of cellular senescence. Nat. Aging 2, 742–755 (2022). source

In the paper

“DBiT-seq is an unbiased multiomic approach that allows for spatial barcoding of the transcriptome in tissues57.”

What the sources say

• Supported

  The Discussion describes DBiT-seq as an unbiased, genome-wide approach that measures the mRNA transcriptome and protein markers on a fixed tissue slide. The Results section (“DBiT-seq workflow”) explains how spatial barcodes are incorporated into tissue-derived cDNAs to reconstruct a spatial expression map. Page numbers are not provided in the supplied passages. (LLM, conf=0.94)

  cites [57] Liu, Y. et al. High-spatial-resolution multi-omics sequencing via deterministic barcoding in tissue. Cell 183, 1665–1681 (2020). source

In the paper

“There are many challenges to mapping senescent cells in tissues such as the absence of specific markers and their relatively low abundance and vast heterogeneity.”

In the paper

“The spatial component is essential, as senescent cells communicate with neighboring cells, impacting their function and the composition of extracellular space.”

In the paper

“Single-cell technologies have allowed unprecedented characterization of senescence; however, many methodologies fail to provide spatial insights.”

In the paper

“There are, however, several advantages: these techniques are established, widely used and inexpensive; as such, protocols are relatively easy to apply and are generally reproducible.”

In the paper

“Factors such as tissue processing and autofluorescence greatly affect image quality and readout accuracy.”

In the paper

“All methods mentioned here have the limitation of detecting one or relatively few biomolecules simultaneously and are therefore not adequate to characterize the molecular heterogeneity of senescent cells in a spatially resolved manner.”

In the paper

“Preparation and preservation of the cell suspension can affect the final proportion of cells that are sampled.”

In the paper

“In immunohistochemical techniques, common challenges in visualizing senescence-associated markers are low levels of antigen expression, lack of specificity of the primary antibody, variability between antibodies even from the same manufacturer, cross-reactivity between primary and/or secondary antibodies, autofluorescence, quality of tissue fixation and others.”

In the paper

“Detection of the activity of the lysosomal enzyme SA-β-Gal at pH 6.0 is a common method used to detect senescent cells20.”

What the sources say

• ?Not checked — no full text of the sourcecites [20] Dimri, G. P. et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367 (1995). source

In the paper

“Limitations of this methodology for identification of senescent cells are that it requires frozen tissue sections (not applicable to formalin-fixed paraffin-embedded (FFPE) samples) and its diffused cytoplasmic staining can be challenging to evaluate.”

What the sources say

• ?Not checked — no full text of the sourcecites [20] Dimri, G. P. et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367 (1995). source

In the paper

“In addition, it is well established within the senescence community that many commercially available antibodies that target mouse p16INK4A are unspecific, making its detection in tissues unreliable.”

In the paper

“Both methods require high-resolution fluorescence imaging, generation of z stacks (to acquire three-dimensional (3D) images) and have high analytical burden and therefore are not amenable to large-scale studies.”

In the paper

“For instance, expression of p16INK4A protein ranges from undetectable to qualitatively low in aged tissues and is an example of a senescence-associated marker that requires a consistent and robust limit of detection sensitivity for accurate studies.”

In the paper

“Image resolution using these systems is similar to that of the aforementioned cyclical staining methods, although fluorescence-based detection may still suffer from high noise-to-signal ratios for low-expression targets such as p16INK4A, particularly in tissues that have undergone harsh fixation.”

In the paper

“Iterative immunofluorescence methods, such as MACSima and CellScape, are capable of imaging with enough resolution to detect subtle changes in organelle structure and morphology (100–200 nm per pixel) and do not require specialized antibody conjugations, which are time consuming and expensive and have the potential to negatively affect their ability to recognize targets.”

In the paper

“However, all iterative immunofluorescence methods share several caveats in their application. Repeated rounds of staining and bleaching may alter epitope stability and are subject to incomplete fluorophore inactivation between cycles.”

In the paper

“Furthermore, mass imaging obliterates the tissue sample, rendering multiplexed ion beam imaging (MIBI) and imaging mass cytometry (IMC) incompatible with sequential analyses.”

In the paper

“Irrespective of staining and detection modalities, platforms that limit the imageable tissue area (CosMx, MACSima, CellScape, PhenoCycler, IMC) may altogether miss populations of senescent cells, which may be unequally distributed, in subsections of larger tissues.”

In the paper

“The majority of current high-plex spatial proteomic platforms use panels of tagged antibodies to detect and quantify between ten and 60 proteins, although some have reported higher numbers.”

In the paper

“This method, however, is technically limiting and expensive.”

In the paper

“Mass spectrometry-based platforms, which use up to 40 different metal-conjugated antibodies in a single staining and detection step, permit highly sensitive protein detection.”

In the paper

“Although Visium is an unbiased spatial transcriptomic solution, its low resolution (100 μm center to center; by comparison, the naked human eye has a resolution of ~40 μm) makes it unsuitable for profiling single cells. Therefore, isolating the senescent cell transcriptome and characterizing their spatial location will not be precise.”

In the paper

“To overcome this limitation, many laboratories have taken advantage of microfluidic systems to image multiple cycles of STORM using the same dye.”

In the paper

“NanoString GeoMx is a spatial omics platform for analysis of RNA (whole transcriptome) or proteins (over 100-plex) that uses photocleavable oligonucleotide barcodes that are coupled to RNA probes or antibodies.”

What the sources say

• ?Not checked — no full text of the sourcecites [56] Merritt, C. R. et al. Multiplex digital spatial profiling of proteins and RNA in fixed tissue. Nat. Biotechnol. 38, 586–599 (2020). source

In the paper

“A solution to overcome this problem is to use conventional widefield microscopy to identify putative senescent cells, which are then targeted for super-resolution imaging.”

In the paper

“In these platforms, the diversity of barcode conjugates makes them unparalleled in their plex limit, potentially exceeding 100 targets on a single tissue section.”

In the paper

“Probes targeting SA-β-Gal will stain the lysosomes specifically, which vary in size from 100 nm to over 1 μm20.”

What the sources say

• ?Not checked — no full text of the sourcecites [20] Dimri, G. P. et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367 (1995). source

In the paper

“Metal tagging can circumvent other issues common to fluorescence-based approaches such as tissue autofluorescence; however, protocols for mass tagging antibodies can negatively impact their sensitivity.”

In the paper

“To date, there has been a disproportionate use of changes in mRNA levels to detect senescent cells.”

In the paper

“High-plex imaging requires substantial time investment in the generation and optimization of conjugated antibody panels, in addition to the time required to acquire images.”

In the paper

“Spatially resolved proteomic approaches have the potential to characterize the density and microanatomical location of senescent cells, to define the impact of senescent cells on their microenvironment and the immune cell types interacting with them and to identify the distribution of key protein senescence effectors within human tissues.”

In the paper

“This is particularly important as it can overcome the gene-throughput limitations of CosMx and STARmap.”

In the paper

“Furthermore, many of the senescence-associated transcripts, such as those encoding cell cycle-dependent kinase inhibitors, are not very abundantly expressed; therefore, high-resolution and high-capture output are essential for spatial analysis of cell senescence.”

In the paper

“Considering that single cells can occupy up to 100–500 μm2, this output is comparable to that of conventional single-cell RNA sequencing (scRNA-seq), enabling sensitive detection of senescent transcripts.”

In the paper

“Such a benchmark is necessary to determine whether methods specifically designed for deconvoluting senescent cells are needed.”

In the paper

“While the accuracy for individual nuclei is difficult to establish in tissues, deep learning predictors can offer overall assessment of senescence in tissues along with differential analysis across ages, medical conditions or other factors.”